Microfluidics is a recent multidisciplinary science, which deals with very small volumes of liquids, from microliters down to femtoliters. Its earliest application concerned inkjet printheads, but it proved to be suitable for the development of “lab-on-a-chip” technology, especially in the field of biotechnology, wherein samples are characterized by very small sizes. Molecular biology, enzymatic analysis, genomic analysis, proteomics, clinical pathology, diagnostics, environmental analysis, etc. all are fields of potential exploitation of microfluidics.
At such microscale dimensions, fluids may show a very different behavior compared to macroscale, a feature which must be taken into account when designing microfluidic devices or experiments which make use of them. For instance, surface tension, energy dissipation, fluidic resistance and diffusion, may largely influence the outcome of the experiments.
For these reasons, ordinary or customary protocols of these assays cannot be directly used in microfluidic devices, but special procedures must be instead designed before implementation.
Advantages linked to the use of microfluidics go back to the easier handling allowed by such devices, the higher flux control permitted, the reduced time of analysis, the high control granted over both concentration and molecular interactions, the incomparable cost saving for reagents and waste products, thus making its use more environmental friendly and giving the ability to process more samples with less space due to reduced instruments hindrance.
The above advantages enable experiments including the use of microfluidic devices to be automated, which would be very interesting from the industrial point of view.
Microchip biotechnology, in particular, is gaining the most from microfluidics, thanks to new developed integrated workflow.
Lab-on-chip devices are few square millimeters to few square centimeters chips on which the bio-assays are reproduced at much smaller scale, in the form of microfluidic circuits.
Lab-on-chip devices, or microfluidic circuits for use in said devices, are widely described in the literature.
U.S. Pat. No. 6,613,560 discloses miniaturized devices for conducting chemical and biochemical processes, in particular a microreactor for conducting DNA amplification; this document faces the problem of unwanted adsorption of the sample under analysis by the walls of the microreactor, and proposes the use of microreactors made (or with surfaces covered) with materials that exhibit reduced adsorption of compounds present in the sample.
International patent application WO 95/22051 discloses a flow cell device having in its channels immobilized reagents which produce an electrically or optically detectable response to an analyte which may be contained in a test sample.
European patent application EP 1542010 discloses a microfluidic device comprising a reaction area, designed to host a reaction between at least a species present in the sample and at least one specific substance, fixed in the area, that can cause interaction specifically or non-specifically with one or more predetermined substances (target species). The secure fixing of the specific substance to the walls of the microfluidic circuit is obtained by means of an intermediate, immobilized film (generally made of an organic compound) previously formed on said walls.
Lab-on-chip devices are already available for use in a variety of analytical techniques, such as electrophoresis, chromatography, staining, fluorescence cytometry, protein analysis, polymerase chain reaction, blood analysis, etc. and, as a further application, Fluorescence In Situ Hybridisation (FISH). As a general reference to FISH, see, for instance, “Cytogenetic and FISH techniques in Myeloid Malignancies”, L. J. Campbell, Methods in Molecular Medicine, 2006, Vol. 125, pp. 13-26.
More in detail, FISH is a very sensitive tool used in diagnostics for the detection of genome alterations.
FISH represents a very promising diagnostic tool for the identification of chromosomal rearrangements or abnormalities, which cannot be detected with other conventional techniques. For example, the analysis of alterations in the chromosomes may be predictive of a future disease or of a therapy response.
As a first step, FISH requires the cell immobilization onto a support, such as, for instance, a microscope glass slide; after that, cells undergo a protein digestion in order to remove cytoplasmic and chromosomal proteins, thus allowing an improved “access” to chromosomal DNA, which needs to be denatured, for example by incubating with formaldehyde-based solutions. After cell dehydration with ethanol-based series of solutions, DNA probes are added. Denaturation is then performed at about 75° C. for 2-5 min and incubation is allowed. A treatment with a suitable post-hybridization solution enables non-specific disturbing bindings due to cross-hybridization to be avoided. Abnormality sites in the chromosome sequence become thus evident by fluorescence imaging.
Prior to FISH, the analysis of DNA made use of scarcely cost-effective methods, while, nowadays, FISH allows researchers to rapidly investigate and understand the basis of many diseases and cancers.
For instance, FISH finds already application in bone marrow testing for haematological tumors, such as leukaemia, lymphoma and myeloma, in solid tumor, lymph node and peripheral blood testing, in preimplantation genetic diagnosis, in prenatal and in postnatal genetic abnormalities screenings.
As a general advantage, FISH may be applied directly to tumor samples, such as biopsies, sections or paraffin-embedded material, providing resolution up to single cell level, enabling the detection of rare events on a suitable cell sample. Despite the potential advantages offered by this technique, its practical adoption has been hindered so far by several drawbacks.
In first place, FISH is extremely expensive, both in terms of reagent costs and of men-time and machine-time necessary to perform the protocol and the image analysis. This limit prevents FISH from being a mass screening method.
A suitable approach to overcome this limit would be the development of a miniaturized protocol by exploiting the features of microfluidic devices.
However a further limit of FISH protocols and devices that can be predicted is the low efficiency of cell adhesion in microfluidic devices; this is due to the fact that, owing to the very limited cross-sections of microfluidic channels, relatively high pressures must be applied to the liquid samples in order to have these moving in the device, which in turn lead to relatively high flow rates. For instance, the article “FISH and chips: chromosomal analysis on microfluidic platforms”, V. J. Sieben et al, IET Nanobiotechnologies, 2007, 1 (3) pp. 27-35, describes a standard adhesion protocol by cytospinning, that however only obtains a yield of retention of the target analytes in the channel of the microfluidic device of 20%. This feature could increase the rate of “false negatives” when searching for rare alterations.
A microfluidic device capable of efficiently immobilizing cells, suitable to perform FISH assays, would help in spreading the use of the technique.
Several prior art documents have the task of improving the retention of analytes in the channels of microfluidic devices.
The paper “Enforced Adhesion of Hematopoietic Cells to Culture Dish Induces Endomitosis and Polyploidy”, X. Huang et al., Cell Cycle, 4(6), pages 801-805, discloses the use of substrates functionalized with poly-D-lysine for enhancing cells adhesion; substrates with a poly-D-lysine coating, commercially available for instance from BD Biosciences, are presently considered the state-of-the-art for cells adhesion and are commonly used in this field of research.
International patent application WO 2008/031228, in the name of the University of Alberta, discloses a fixating protocol that allows to reach a percentage of adhered cells up to 75% of the total. The immobilization of cells in the microfluidic channels is obtained by raising the temperature in the range between 50 and 95° C. for a period of time, determined by intervention of a human operator, sufficient to allow immobilization of a portion of a population of cells of interest. As a consequence, despite the improvement in the percentage of adhered cells, the method of this document still suffers from the limits that the immobilization step must be controlled by a human operator, and that the relatively high temperatures needed in this step could damage some cells. Besides, the application describes two embodiments of microfluidic device, called “Microchip” and “Circulating Microchip”, respectively. The embodiment named “Microchip” is made of a 0.5 mm thick microscope glass slide carrying the microfluidics and a coverslip 0.17 mm thick, declared to be necessary to create a minimum working distance for high resolution imaging. Both the device components are extremely fragile and require an extremely careful handling during assembling, preventing an easy scale up of the device in industrial settings. The embodiment named “Circulating Microchip” is made of two 1.1 mm thick microscope glass slides and of a middle PDMS layer 0.254 mm thick. This embodiment overcomes the fragility problems of “Microchip”, but its thickness does not allow the use of a 100× lens for the image acquisition, thus preventing to obtain high resolution images, as required by current FISH standards.
European patent application EP 1215186 discloses a support, said to be useful for immobilizing oligonucleotides, that can be used in the fabrication of microfluidic devices; this support has the surface functionalized with an oxide chosen among HfO2, TiO2, Ta2O5, ZrO2 and their mixtures, treated after their deposition in order to make their surface hydrophilic. This document is silent however about the immobilization of cells.
International patent application WO 00/33084 discloses a wide range of devices for use in diagnostics, in which the active surface is functionalized with organic compounds, possibly laid over a “gelled network” oxide. This document does not give any information about actual retention yields of cells.
Accordingly, a FISH device and method which would overcome the disadvantages of the prior-art methods, both classic and microfluidic, is needed.
In particular, it would be desirable to design a device and a process, which would be cheap, easy to handle, scale up and perform, fast to be carried out and efficient as well.